Purinergic Signalling (2017) 13:489–496

DOI 10.1007/s11302-017-9575-2

ORIGINAL ARTICLE

Diphenyl diselenide modulates nucleotidases, reducing

inflammatory responses in the liver of Toxoplasma gondii-infected
mice
Pedro H. Doleski 1,2 & Daniela B. R. Leal 1 & Vanessa S. Machado 1 & Nathieli B. Bottari 2 &
Alessandra G. Manzoni 1 & Emerson A. Casali 3,4 & Cesar E. J. Moritz 3,5 &
Ana C. A. Rocha 3 & Giovana Camillo 6 & Fernanda F. Vogel 6 & Lenita M. Stefani 7 &
Ricardo E. Mendes 8 & Aleksandro Schafer da Silva 2,7

Received: 10 February 2017 / Accepted: 21 July 2017 / Published online: 16 August 2017
# Springer Science+Business Media B.V. 2017

Abstract The aim of this study was to verify the effect of
diphenyl diselenide (PhSe)2 on hepatic nucleotidases and on
the concentration of purines in mice infected by Toxoplasma
gondii. The animals were divided into four groups: Group A
(uninfected), Group B (uninfected and treated with (PhSe)2),
Group C (infected), and Group D (infected and treated with
(PhSe)2). The inoculation (groups C and D) was performed
with 50 cysts of T. gondii (ME-49 strain). Mice from groups B
and D were treated with 5 μmol kg−1 of (PhSe)2. Liver tissue
from infected mice showed less severe inflammation, elevated
ATP/ADO ratio, elevated NTPDase, 5′nucleotidase, and ADA
* Aleksandro Schafer da Silva
aleksandro.silva@udesc.br
1
Department of Microbiology and Parasitology, Universidade Federal
de Santa Maria (UFSM), Santa Maria, Brazil
2
Department of Biochemistry and Molecular Biology, Universidade
Federal de Santa Maria (UFSM), Santa Maria, Brazil
3
Department of Morphological Science, Universidade Federal do Rio
Grande do Sul (UFRGS), Instituto de Ciências Básicas da Saúde
(ICBS), Porto Alegre, Brazil
4
Department of Biochemistry, Universidade Federal do Rio Grande
do Sul (UFRGS), Instituto de Ciências Básicas da Saúde (ICBS),
Porto Alegre, Brazil
5
Program of Medical Science, Universidade Federal do Rio Grande do
Sul (UFRGS), Instituto de Ciências Básicas da Saúde (ICBS), Porto
Alegre, Brazil
6
Department of Preventive Veterinary Medicine, Universidade
Federal de Santa Maria (UFSM), Santa Maria, Brazil
7
Department of Animal Science, Universidade do Estado de Santa
Catarina (UDESC), Chapecó, SC, Brazil
8
Laboratory of Veterinary Pathology, Instituto Federal Catarinense
(IFC), Concórdia, SC, Brazil
activities compared to the uninfected group (Group A;
P < 0.05). However, infected and treated mice showed de-
creased ATP levels and elevated ADO levels, as well as higher
NTPDase and 5′nucleotidase activities and decreased ADA
activity in the hepatic tissue compared to the infected group
(P < 0.05). Moreover, the (PhSe)2 treatment of infected mice
reduced the hepatic inflammation and showed an immuno-
modulatory effect on ectonucleotidases of hepatic lympho-
cytes, which it returned to basal levels. Therefore, chronic
infection by T. gondii induces hepatic inflammation in mice,
and it is possible that purine levels and nucleotidase activities
in hepatic tissue are related to the pathogenesis of the infection
in this tissue. The treatment with (PhSe)2 was able to reverse
the hepatic inflammation in mice chronically infected, possi-
bly due to the modulation of purinergic enzymes that produce
an anti-inflammatory profile through the purinergic system in
the liver tissue.
Keywords Diphenyl diselenide . Toxoplasma gondii .
Hepatic lymphocytes . Purines . Nucleotidases
Introduction
Toxoplasmosis is a zoonosis caused by Toxoplasma gondii,
and felines are the definitive hosts. They may release oocysts
of the parasite in the environment, contaminating accidental
hosts, as humans and animals [1]. Usually healthy infected
hosts do not show clinical signs, but this depends on the host
immune response and also the strain of the parasite. The ME-
49 strain is known for causing chronic toxoplasmosis, in
which the parasite causes the latency form of the disease in
the brain tissue [2]. However, studies demonstrated an
490
association between infection and hepatic inflammation that
leads to cirrhosis [3]. The liver plays an important role in
controlling many metabolic routes of the body [4]. For pro-
tection, the organ has distinct immune cells such as Kupffer
and stellate cells and lymphocytes responsible for the mainte-
nance of the immune response in the tissue. A health liver has
approximately 1010 lymphocytes to protect the organ by
responding to innumerable inflammatory mediators that in-
duce cytokine release and activation and recruitment of new
immune cells [5]. Hepatic pathologies increase the number of
lymphocytes that are activated through inflammatory signal-
ing, which induce these cells to release pro-inflammatory cy-
tokines, changing the immune response of this organ [6].
Besides cellular effectors, the immune system has numerous
chemical mediators to perform the signaling of the immune
response in the body [7].
The purinergic signaling is characterized by extracellular
purine nucleotide interactions with specific cell receptors ca-
pable of modulating hepatic functions, leading to cell prolif-
eration and vascularization, acting as an immune mediator [8].
When ATP interacts with receptor type P2, it triggers an in-
crease in hepatocyte proliferation, causing vasodilation in si-
nusoidal vessels and enabling the immune response [9]. On
the other hand, when the ATP degradation product adenosine
(ADO) interacts with receptor type P1, it induces the release of
anti-inflammatory cytokines, enhancing the collagenolytic ac-
tivity in the liver. In this way, ADO exerts an anti-fibrotic
effect and develops a protective cellular environment [9, 10].
Adenosine has a fundamental function in homeostasis of
healthy tissue. However, low concentration of this molecule
is related to the development of chronic liver diseases [11].
Healthy hepatic cells release low ATP concentrations
into the extracellular medium through membrane chan-
nels; however, this release is increased after stimuli, such
as the interaction with inflammatory mediators, stress, and
cell damage. While low ATP levels have important phys-
iological functions, high extracellular concentration of it
can act as damage-associated molecular patterns
(DAMPs), inducing pro-inflammatory events through
lymphocyte activation [12]. Due to these events, the nu-
cleotide concentration in the extracellular space is regu-
lated by nucleotidases, enzymes expressed on a soluble
form in the interstitial tissue or bound in the plasma mem-
brane of the cells, so-called ecto-enzymes [13]. This reg-
ulation starts with NTPDase that hydrolyses ATP and
ADP into AMP. The second phase occurs by 5′nucleotid-
ase, which hydrolyses AMP into Ado. Adenosine deami-
nase (ADA) is the third regulatory enzyme that deami-
nates Ado into inosine. Nucleotidase enzymes regulate
nucleotide interactions through the increase or decrease
of its concentration in the medium, and countless studies
have shown the importance of this enzymatic regulation
in physiological and pathological processes [14, 15].
Purinergic Signalling (2017) 13:489–496
Diphenyl diselenide (PhSe)2 is an organoselenium com-
pound with antioxidant, antiviral, and immunomodulatory ac-
tivities [16]. Several in vivo experiments show that (PhSe)2
reduces the levels of pro-inflammatory cytokines and reactive
species in various pathologies; however, the true mechanism
for its protective and anti-inflammatory action is still not fully
understood [17, 18]. Studies indicated that (PhSe)2 reacts with
thiol groups in the tertiary protein structure, and this interac-
tion modulates the enzymatic activity and affinity to sub-
strates, causing alterations on important metabolic pathways,
and this is possibly the molecular mechanism of action [19].
In the chronic phase of toxoplasmosis of immuno-
competent mice, the parasite is latent in the brain, but
the infection is still able to induce damage to other
tissues such as the liver. It is possible that components
of the purinergic system are involved in the pathology
of T. gondii infection, whereas the damage caused to the
body is due to a pro-inflammatory signaling in non-
infected tissues. Therefore, the aim of this study was
to investigate the concentration of purines and the ac-
tivity of nucleotidases in hepatic tissue of mice infected
by T. gondii treated with diphenyl diselenide (PhSe)2, as
well as the effect of this treatment on ectonucleotidase
activity in hepatic lymphocytes, which act as a biomark-
er of the immune response.
Materials and methods
Chemicals
The substrates ATP, ADP, AMP, adenosine, Coomassie Blue,
(PhSe)2, and Ficoll-Hypaque (1.077 g/mL) were obtained
from Sigma Chemical Co. (St. Luis, MO, USA). All other
reagents used in the experiments were of analytical grade with
high purity.
Study design
Forty 60-day-old Swiss albino mice, weighing approximately
25–30 g, were used in the experiments. The T. gondii
cystogenic strain (ME-49) used was maintained by consecu-
tive passages in the brain of mice every 30 days. The animals
were divided into four groups with 10 mice each: Group A
(uninfected), Group B (uninfected treated with (PhSe)2),
Group C (infected), and Group D (infected treated with
(PhSe)2). Twenty mice (groups C and D) were infected orally
with 250 μL of brain homogenate containing 50 parasitic
cysts of T. gondii. Mice from groups B and D were treated
on days 1 and 20 post-infection (PI) with 5 μmol kg−1 of
(PhSe)2 by subcutaneous injection [19].
Purinergic Signalling (2017) 13:489–496
Sampling
Thirty days PI, all animals were anesthetized in an isoflurane
chamber. For seric markers, blood samples were collected by
cardiac puncture without anticoagulant, and sera samples were
obtained after centrifugation at 3000 rpm for 15 min. Livers
were removed and divided into four parts with approximately
3 g each. Two parts were used to histopathology and lympho-
cyte isolation. Another part was used for the determination of
purine levels, and for that, the sample was gently homoge-
nized in sterile flasks, and the resultant supernatant was mixed
with 0.6 M perchloric acid and 1 M potassium hydroxide, as
described previously [20]. And, the last fragment was manu-
ally homogenized in 10 mL of physiological solution (PS)
with a syringe plunger and centrifuged at 1500 rpm for
15 min. The supernatant was removed and used for hepatic
lymphocyte isolation. Protein was measured by the
Coomassie Blue method according to Bradford [21] using
serum albumin as the standard.
Histopathology
At the necropsy, tissue samples were collected from the right
hepatic lobes, fixed in 10% buffered formalin in 0.1 M phos-
phate buffer. The fixed tissue was dehydrated and embedded
in paraffin. Tissue sections were stained with hematoxylin and
eosin (H&E) for histopathological examination.
Seric markers of hepatic damage
Seric activities of alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), and lac-
tate dehydrogenase (LDH) were evaluated using commercial
kits (Analisa®).
Hepatic purine levels
ATP and its breakdown products were analyzed by HPLC
according to Voelter et al. [21]. The proteins were
denaturated using 0.6 mol/L of perchloric acid. All samples
were centrifuged (16,000×g for 10 min at 4 °C); superna-
tants were neutralized with 4.0 N KOH, and clarified with a
second centrifugation (16,000×g for 30 min at 4 °C). After
the second centrifugation, the supernatants were collected
and centrifuged again (16,000×g for 30 min at 4 °C).
Aliquots of 20 μL were applied to a reversed-phase HPLC
(LC-20AT model, Shimadzu, Kyoto, Japan) using a C18
column (Ultra C18, 25 cm × 4.6 mm × 5 μm, Restek,
USA). The elution was carried out applying a linear gradi-
ent from 100% of solvent A (60 mM KH2PO4 and 5 mM of
tetrabutylammonium chloride, pH 6.0) to 100% of solvent
B (solvent A plus 30% methanol) over a 30-min period
(flow rate at 1.4 mL/min). Mobile phases were filtered
491
through a 0.22-μm Millipore filter prior to analysis, and
all the reagents were of HPLC grade. The amount of purines
and metabolic residues were measured by absorption at
254 nm. The retention time of the standards was used as a
parameter for identification and quantification by compar-
ison of the peak area. Purine levels were expressed as nmol
of different compounds per g of tissue.
Isolation of hepatic lymphocytes
Hepatic lymphocytes were isolated from approximately 3 g of
liver tissue carefully homogenized in PBS (phosphate buffer
solution) and separated on Ficoll-Hypaque density gradients
as previously described by Doleski et al. [22].
E-NTPDase activity in hepatic lymphocytes
Activity of E-NTPDase in hepatic lymphocytes was previ-
ously described by Doleski et al. [22]. Twenty microliters
of intact lymphocytes were added to the reaction medium
containing 50 mM Tris–HCl buffer (pH 8.0), supplemented
with 0.5 mM CaCl2, 120 mM NaCl, 5 mM KCl, and 60 mM
glucose in a final volume of 200 μL. The reaction was
initiated by the addition of the substrate (ATP or ADP) at
a final concentration of 2.0 mM in 37 °C and stopped after
40 min of incubation with 200 μL of 10% trichloroacetic
acid (TCA). The samples were chilled on ice, and the
amount of Pi released was measured by the method of
Chan et al. [23]. Enzymatic activities were expressed as
nmol of Pi/min/mg protein.
NTPDase and 5′nucleotidase activities in liver
homogenates
NTPDase and 5′nucleotidase activities in liver homoge-
nates were determined using the method described by
Rosemberg et al. [24], modified by Doleski et al. [25].
First, liver homogenates were centrifuged at 2500 rpm for
10 min to remove possible impurities. The reaction mixture
for NTPDase activity contained 1 mM of ATP or ADP as
substrate, 5 mM CaCl2, and 50 mM Tris–HCl (pH 8.0). For
5′nucleotidase, the reaction mixture contained 10 mM of
AMP as substrate, 5 mM MgCl2, and 50 mM Tris–HCl
(pH 7.5). The reaction mixtures were incubated with ap-
proximately 1.0 mg of homogenized protein at 37 °C for
30 min on a final volume of 0.2 mL. The reaction was
stopped by the addition of 200 μL of 10% TCA. The sam-
ples were chilled on ice and the amount of Pi liberated was
measured by the green malaquite method [23]. Enzymatic
activities were expressed as nmol of Pi/min/mg protein.
492
E-ADA activity in hepatic lymphocytes and homogenates
E-ADA activities were measured spectrophotometrically,
based on the direct formation of ammonia produced when
the enzyme acts on adenosine [26]. The reaction initiated by
the addition of the substrate (adenosine) to a final concen-
tration of 21 mM/L and incubated for 60 min at 37 °C with
25 μL of liver homogenates or intact hepatic lymphocytes.
A E
B F
C G
D H
Fig. 1 Mice experimentally infected by Toxoplasma gondii: Group A
(uninfected), Group B (uninfected and treated with (PhSe)2), Group C
(infected), and Group D (infected and treated with (PhSe)2). Normal
architecture in the brain of mice of groups A (photomicrography—a)
and B (Photomicrography—b) showing the corticomedullary region.
HE, obj. ×10. In the brain of mice from group C, it was observed
intense multivalent mononuclear perivascular cuff (arrowhead)
associated with rounded, encapsulated, and eosinophilic structure filled
with small granules morphologically compatible with cysts of T. gondii
(arrow). Note an extensive area of malacia associated with moderate
mononuclear inflammatory infiltrate (asterisk) HE, obj. ×10
(photomicrography—c). In mice of group D, a cyst containing
bradyzoites was observed in the interior, morphologically compatible
with T. gondii (arrow), associated with mononuclear inflammatory
Purinergic Signalling (2017) 13:489–496
The reaction was stopped by the addition of 1.5 mL of 106/
0.16 mM phenol–nitroprusside to the reaction mixture,
which was immediately mixed with 1.5 mL of 125/11 mM
alkaline-hypochloride (sodium hypochlorite). Released
ammonia reacts with alkaline-hypochlorite and phenol in
the presence of a catalyst-sodium nitroprusside to produce
indophenol (a blue color), and the concentration of ammo-
nia is directly proportional to the absorbance of indophenol
I
J
K
L
infiltrate adjacent to the cyst, HE, obj. ×20 (photomicrography—d).
Liver with normal architecture in animals of groups A
(photomicrography—e) and B (photomicrography—f), HE, obj. ×10.
All mice from Group C showed periportal mononuclear inflammatory
infiltrate in the liver, HE, obj. ×10 (photomicrography—g). In Group D,
mice showed mononuclear inflammatory infiltrate and adjacent
neutrophils (arrow) which corresponds to 40% of the animals in this
group, HE, obj. ×10 (Photomicrography—h). Liver with normal
architecture in animals of groups A (photomicrography—i) and B
(photomicrography—j), HE, obj. ×20. Liver of animals from Group C
showed periportal mononuclear inflammatory infiltrate, HE, obj. ×20
(photomicrography—k). Liver with normal architecture in animals of
group D (photomicrography—l), which corresponds to 60% of the
animals in this group, HE, obj. ×10
Purinergic Signalling (2017) 13:489–496 493

Table 1 Seric enzymatic activity of hepatic damage markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase
(ALP), and lactate dehydrogenase (LDH)) in mice infected by Toxoplasma gondii and treated with diphenyl diselenide (PhSe)2a

Markers Group A Group B Group C Group D P

ALT (U/L) 49.20 ± 2.261 53.74 ± 3.652 70.20 ± 6.948 67.89 ± 8.237 >0.05
AST (U/L) 146.40 ± 16.78 178.70 ± 16.01 189.50 ± 25.44 177.80 ± 13.84 >0.05
LDH (mg/dL) 978.2 ± 117.5 1544 ± 188.7 1420 ± 206.4 1026 ± 95.59 >0.05
ALKP (U/L) 112.0 ± 10.41 150.3 ± 19.45 127.8 ± 26.06 107.7 ± 13.88 >0.05

Results are expressed as mean ± SEM (n = 10). Data were analyzed statistically by two-way analysis of variance (ANOVA). Post-hoc comparisons were
made using Tukey test, and differences were considered significant at P < 0.05. Note: Group A (uninfected), Group B (uninfected and treated with
(PhSe)2), Group C (infected), and Group D (infected and treated with (PhSe)2)

read at 620 nm. Ammonium sulfate of 75 μM was used as Seric markers of hepatic damage
ammonium standard. The value of E-ADA activity was
expressed as U/mg of protein. Seric AST, ALT, LDH, and ALP activities did not show sig-
nificant difference between groups by two-way ANOVA
(Table 1).
Statistical analysis

All experiments were performed in triplicate. Data represent
mean ± standard deviation. Differences were assessed by two-
way analysis of variance (ANOVA), followed by Tukey post-
hoc test. The values obtained in the assays were considered
statically different for P < 0.05.
Results
Histopathology
Animals infected by the strain ME-49 of T. gondii showed
bristling hair and weight loss (groups C and D), and the infec-
tion was confirmed by histopathology, i.e., there were parasit-
ic cysts in the brain of all infected animals in both groups.
Mice of groups A and B did not show histological lesions in
the liver. All mice of Group C showed mild inflammatory foci
in the liver (Fig. 1), and alterations of this kind were observed
in only four mice of Group D.
Hepatic purine levels
The levels of purine in liver samples are shown in
Table 2. The (PhSe)2 treatment (Group B) caused an
increase of 60.8% on ATP concentrations and a decrease
of 56% in ADP concentrations in the liver. Moreover,
AMP levels did not show difference compared to the
control group (Group A), but interestingly, there was
an increase of 236% in Ado concentrations. Infected
group (Group C) showed a great increase (150%) in
ATP concentration and a decrease of 84.6% for ADP,
21% for AMP, and 51.2% for Ado. (PhSe)2 treatment
used in infected mice (Group D) decreased (24.3%)
ATP concentration compared to infected mice (Group
C), but when compared to the control group (Group
A), it showed an increase of 88.8%. Group D showed
an increase of 35% for AMP and 81.2% decrease of
ADP levels, while Ado concentration did not show sig-
nificant difference compared to the control or infected
groups.

Table 2 Purine concentrations in

the liver of mice infected by Purines Group A Group B Group C Group D
Toxoplasma gondii and treated
with diphenyl diselenide (PhSe)2a ATP (nmol/g) 1.43 ± 0.67c 2.30 ± 0.93b 3.57 ± 0.47a 2.70 ± 0.23b
ADP (nmol/g) 10.2 ± 3.41a 4.49 ± 1.87b 1.57 ± 0.78c 1.92 ± 1.02c
AMP (nmol/g) 136.3 ± 25.7b 132.1 ± 28.4b 107.5 ± 14.9c 184.1 ± 45.4a
Adenosine (nmol/g) 19.8 ± 4.8b 66.5 ± 14.9a 9.66 ± 3.7c 13.4 ± 5.1bc

Results are expressed as the mean ± SEM (n = 10). Data are analyzed statistically by two-way analysis of variance
(ANOVA). Post-hoc comparisons were made using Tukey test, and differences were considered significant at a
P < 0.05. Groups with equal letters in the same line do not differ statistically. Note: Group A (uninfected), Group B
(uninfected and treated with (PhSe)2), Group C (infected), and Group D (infected and treated with (PhSe)2)
494
Fig. 2 Mice infected by Toxoplasma gondii and treated with diphenyl
diselenide (PhSe)2. Hepatic lymphocyte E-NTPDase activity to ATP (a)
and ADP (b) substrates. Columns represent mean ± SEM (n = 10).
Different letters in the same graph denote significance for P < 0.05 by
two-way ANOVA followed by Tukey post-hoc test. Note: Group A
(uninfected), Group B (uninfected and treated with (PhSe)2), Group C
(infected), and Group D (infected and treated with (PhSe)2)
E-nucleotidases in hepatic lymphocytes
ATP and ADP hydrolysis by E-NTPDase in plasma mem-
brane of hepatic lymphocyte samples are shown in Fig. 2. E-
NTPDase activity for ATP substrate decreased 22%, and it did
not show significant difference for ADP substrate in Group B.
Animals of Group C showed an increase of 49.5% and 56.6%
in E-NTPDase activity for ATP and ADP compared to unin-
fected animals (Group A), respectively. Mice of Group D did
not show significant differences on E-NTPDase activity for
Fig. 3 Mice infected by Toxoplasma gondii and treated with diphenyl
diselenide (PhSe)2. Hepatic lymphocyte E-ADA activity to adenosine
(Ado) substrate. Columns represent mean ± SEM (n = 10). Different
letters in the same graph denote significance for P < 0.05 by two-way
ANOVA followed by Tukey post-hoc test. Note: Group A (uninfected),
Group B (uninfected and treated with (PhSe)2), Group C (infected), and
Group D (infected and treated with (PhSe)2)
Purinergic Signalling (2017) 13:489–496
both substrates (ATP and ADP) compared to the control group
(Group A).
E-ADA activity in the plasma membrane of lymphocytes is
shown in Fig. 3. Mice of Group B did not show significant
difference for E-ADA activity compared to Group A, but the
infected group (the Group C) showed a decrease of 70.6% in
this enzyme. Group D (infected and treated with (PhSe)2) did
not show a significant difference compared with Group A.
Hepatic nucleotidases
Nucleotidase activities in liver homogenates are shown in
Table 3. NTPDase activities in Group B showed an increase
of 46.52 and 46.27% to ATP and ADP hydrolysis, respective-
ly. There was an increase of 26% to AMP hydrolysis by 5′
nucleotidase activity and a decrease of 44.4% in ADA activity
compared to the control (Group A). Infected animals (Group
C) showed increased nucleotidase activities such as NTPDase
(37.3 and 35.2% to ATP and ADP hydrolysis, respectively)
and 5′nucleotidase (44.8% to AMP hydrolysis), and ADA
activity (50.5%) compared to Group A. However, (PhSe)2
treatment of infected mice (Group D) increased NTPDase
activity (93.2 and 89.6% in ATP and ADP hydrolysis, respec-
tively), as well as 5′nucleotidase activity (57.6%). ADA activ-
ity did not show significant differences compared to uninfect-
ed (Group A) or infected animals (Group C).
Discussion
T. gondii infection was confirmed by histopathological exam-
inations of the brain that showed parasitic cysts with
bradyzoites. These findings are common for T. gondii ME-
49 strain, since it develops chronic toxoplasmosis during the
parasitic latent phase [1, 2]. Histological examinations of the
liver of infected animals revealed a slight inflammation and
(PhSe)2 treatment it in 60% of the animals. Serological anal-
yses for markers of hepatic injury (ALT, AST, LDH, ALP) did
not confirm tissue damage, possibly due to a slight inflamma-
tory process histologically found, which did not cause cell
lysis, and consequently, the release of such markers in the
bloodstream.
Firstly, it is worthy to note that (PhSe)2 is a Janus-faced mol-
ecule, so its pharmacological effect or toxicity depends on the
concentration used. Treatment with (PhSe)2 in healthy mice
caused ATP increase compared to healthy untreated animals.
The mechanism to explain this result is unknown; however, it
is possible that the (PhSe)2 stimulates a constant release of ATP
by hepatic cells, and by a compensatory effect, it raises hepatic
NTPDase activity in order to control the levels of this phosphate
nucleotide. The powerful anti-inflammatory action of (PhSe)2
was confirmed on the purinergic system by a significant increase
in adenosine concentration in liver tissue, an important
Purinergic Signalling (2017) 13:489–496 495

Table 3 Hepatic NTPDase

(nmol of Pi/min/mg protein), 5′ Nucleotidases Group A Group B Group C Group D
nucleotidase (nmol of Pi/min/mg
protein), and ADA (U/mg of NTPDase activity
protein) activity in infected mice ATP substratum 5.341 ± 0.735c 7.826 ± 0.555b 7.334 ± 0.178b 10.320 ± 0.420a
by Toxoplasma gondii and treated ADP substratum 5.469 ± 0.572c 8.003 ± 0.559b 7.395 ± 0.256b 10.370 ± 0.354a
with diphenyl diselenide (PhSe)2
a 5′nucleotidase activity
AMP substratum 5.362 ± 0.203c 6.763 ± 0.428b 7.766 ± 0.127ab 8.449 ± 0.143a
ADA activity
Ade substratum 1.285 ± 0.108b 0.714 ± 0.093c 1.934 ± 0.079a 1.641 ± 0.190ab

Results are expressed as mean ± SEM (n = 10). Data were analyzed statistically by two-way analysis of variance
(ANOVA). Post-hoc comparisons were made using Tukey’s test and difference were considered significant at a
P < 0.05. Groups with letters equal the same line not statistically different. Note: Group A (uninfected), Group B
(uninfected and treated with (PhSe)2), Group C (infected), and Group D (infected and treated with (PhSe)2)

endogenous anti-inflammatory molecule [14], which may be as-
sociated with the removal of inflammatory infiltrates in liver
found in the majority of treated animals. The mechanism for this
effect was associated with an increase in 5′nucleotidase activity
and decrease in the ADA activity, stimulating the production and
inhibition of adenosine degradation. It is known that the (PhSe)2
is able to interact with thiol groups, altering many metabolic
processes, including enzymatic activity and its affinity for sub-
strates, which can act as a possible pharmacological mechanism
[19]. Our hypothesis is that the (PhSe)2 acts by two ways: (1)
increasing the concentration of nucleotides, such as ATP, and
modulating by a compensatory manner the enzymatic activity
or (2) interacting covalently with the enzymes in question, pro-
ducing an anti-inflammatory effect through the purinergic signal-
ing pathway. To evaluate the effect of (PhSe)2 on modulating the
immune response, ectonucleotidases were evaluated in hepatic
lymphocytes since they act as biomarkers of the immune re-
sponse, i.e., enzymatic activity in these cells depends on the
immune microenvironment. Liver lymphocytes showed a low
decrease in the E-NTPDase activity after (PhSe)2 treatment of
healthy mice, possibly by stimulating the interaction of ATP with
P2 receptors, enhancing the immune activity in a controlled man-
ner, which demonstrates an immunomodulatory effect of (PhSe)2
[12].
Infected mice showed the highest concentration of ATP,
and it is known that high ATP levels are usually presented
as a DAMP related to the inflammatory process in the tissue
[9]. It is possible that mediators present after T. gondii in-
fection stimulate a cellular release of ATP by transport
channels, in order to perform a local pro-inflammatory sig-
naling. In an attempt to reduce high ATP levels, the tissue
shows a large increase in NTPDase activity, although it is
still unable to reduce exposure to the nucleotide. It is also
possible to assure that T. gondii infection may increase all
nucleotidases analyzed in this study, such as ADA and 5′
nucleotidases, which in turn eventually reduces the concen-
tration of AMP and Ado in the tissue. The nucleoside Ado
acts to protect and reverse liver damage, but low interaction
of this molecule with the P1 receptor is associated with the
development of chronic liver diseases [10]. Ectonucleotidase
activity in lymphocytes helps to understand this complex
mechanism, since increased E-NTPDase activity controls high
ATP interaction with P2 receptors in the cell membrane, and
decreased E-ADA activity stimulates the interaction of low
Ado concentration with its P1 receptors. In this way, lympho-
cytes try to avoid an exacerbated pro-inflammatory activation
triggered by ATP.
Interestingly, (PhSe)2 treatment of infected mice showed a
reduction in ATP concentration in liver homogenates. We pro-
pose that this effect is related to the high NTPDase activity
found in the hepatic tissue of this group, which is enhanced by
(PhSe)2 compared to infected untreated mice and, consequently,
reduced the nucleotide levels in the tissue. Furthermore, the
consecutive increase in NTPDase and 5′nucleotidase activities
along with decreased ADA activity in the tissue resulted in
increased adenosine concentration, and possibly, increased inter-
action with P1 receptors, which may protect the hepatic tissue.
This low-ratio ATP/adenosine helps to understand the anti-
inflammatory mechanism produced by (PhSe)2, which is known
to produce many benefits in vivo [16]. (PhSe)2 used to treat
T. gondii infection was also able to modulate ectonucleotidase
in lymphocytes to baseline levels, and thus, this fact can be
interpreted as a reduction in markers of the immune response
due to the re-establishment of the extracellular environment of
nucleotides.
Our results demonstrated a change in some components of the
purinergic signaling system in the liver of T. gondii-infected
mice, as well as signs of healing through subcutaneous treatment
with (PhSe)2. These results help to understand that some mech-
anisms involved in fighting the infection can be dangerous to the
body itself, where the high ratio of ATP/adenosine found in
infected mice elucidates one possible mechanism that leads to
chronic liver disease in patients with chronic toxoplasmosis. We
emphasize that (PhSe)2 treatment has high capacity to elevate
adenosine concentration in healthy liver tissue, as well as to re-
induce a healthy extracellular nucleotide environment in an
496
inflammatory process. For this, (PhSe)2 is a potential drug that
can be used to treat chronic liver diseases; however, more studies
should be conducted to evaluate its clinical efficacy for chronic
diseases.
Compliance with ethical standards
Conflicts of interest Pedro H. Doleski declares that he has no conflict
of interest.
Daniela B.R. Leal declares that she has no conflict of interest.
Vanessa S. Machado declares that she has no conflict of interest.
Nathieli B. Bottari declares that she has no conflict of interest.
Alessandra G. Manzoni declares that she has no conflict of interest.
Emerson A. Casali declares that he has no conflict of interest.
Cesar E.J. Moritz declares that he has no conflict of interest.
Ana C.A. Rocha declares that she has no conflict of interest.
Giovana Camillo declares that she has no conflict of interest.
Fernanda F. Vogel declares that she has no conflict of interest.
Lenita M. Stefani declares that she has no conflict of interest.
Ricardo E. Mendes declares that he has no conflict of interest.
Aleksandro Schafer da Silva declares that he has no conflict of
interest.
Ethical approval This study was approved by the Ethic Committee on
Animal Use of the Federal University of Santa Maria (UFSM) under
protocol number 7787270815.
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